Cytosine methylation is a DNA modification found in the genomes of eukaryotes including mammals, higher. plants, and filamentous fungi. Methylation is associated with loss of gene expression (silencing) and suppression of homologous recombination. Maintenance of correct methylation patterns is critically important for development. For example, in humans, alteration of methylation patterns in certain genomic regions leads to a number of genetic diseases including mental retardation syndromes and pre-disposition to malignancies. In mice, a cytosine methyltransferase knock-out mutation results in embryo-lethality. In the higher plant model organism Arabidopsis thaliana, methylation-deficient mutants display a number of aberrant morphologies but are nonetheless viable and fertile. Therefore, Arabidopsis provides an extremely tractable system to study the mechanisms of methylation and silencing with molecular and genetic approaches. Our research focuses on a family of methylated endogenous genes in Arabidopsis that consists of two singlet genes plus a tail-to-tail inverted repeat gene duplication at three unlinked genomic positions. Previous work showed that the inverted repeat locus triggers methylation of the identical sequences elsewhere in the genome. Paradoxically, although methylation causes silencing of a singlet locus, a gene in the methylated inverted repeat is expressed. The goal of this proposal is to understand the mechanisms by which the inverted repeat locus escapes the normally repressive effects of methylation. This question will be explored by characterizing the chromatin structure, cis-acting DNA sequences, and trans-acting factors that control methylation and expression of the silenced singlet locus versus the expressed inverted repeat locus.